496
Anal. Chem. 1985, 57,496-500
Sensitive Bioaffinity Sensor with Metastable Molecular Complex Receptor and Enzyme Amplifier Yoshihito I k a r i y a m a , Makoto F u r u k i , and Masuo Aizawa* Institute of Materials Science, University of Tsukuba, Sakura-mura, Ibaraki 305, J a p a n
A unique biosenslng method which uses a molecular complex receptor of low affinity and an enzyme ampllflcation technique Is described for the determination of small molecular substances of physiological importance. The molecular complex, prepared from a membrane-bound analogue compound and a binding protein, dissociates upon exposure to a determinant in solution, since the complex Is a metastable one. The displacement of the analogue compound by a determinant In a given protein binding reaction relates to the bloaffinity difference between an analogue compound and a determinant. The dissociation of the metastable complex receptor and the resulting stable complex formation depend on determlnant concentration. High sensttivity is accomplished by means of an enzyme amplification technique. This concept has been demonstrated by the usage of membrane-bound HABA (or lipoic acid) as an analogue compound, avldln as a blnding protein, and catalase as an enzyme amplifier. Biotln (vitamin H) was sensitively determined in the concentration range from lo-' to lo-' g/mL. The midpoint of the calibration curve was lo-' g/mL. I n the case of n = 10 at the mldpoint concentrailon, coefficient of varlation (CV) was less than i l l %.
Enzymes, antibodies, and binding proteins bind their corresponding counterparts through noncovalent forces with high specificity. Both radioimmunoassay and enzyme immunoassay use the specificity of these protein molecules (1-3). The present paper describes a new sensing principle with high sensitivity based on bioaffinity difference between two ligands, i.e., one a determinant and the other an analogue compound in a given binding reaction. In general an analogue compound shows lower affinity to the binding protein than a determinant does. Therefore, one can expect the following displacement reaction when a membrane-bound analogue compound complexed with its binding protein is exposed to a determinant molecule. The binding protein is displaced from the membrane-bound analogue molecule by the biotin to form a biotin-avidin complex in solution. The displacement is supposed to depend on the bioaffinity difference between an analogue molecule and a determinant as well as the determinant concentration. The determinant concentration may be easily measured by the detection of residual molecular complex remaining on a membrane surface. High sensitivity will be attained by chemical amplification by the usage of an enzyme catalyst as a label. Avidin, an egg white protein, forms a very stable complex with vitamin H (biotin). This protein also binds analogue compounds of biotin such as 2- [ (4-hydroxypheny1)azolbenzoic acid [HABA] and lipoic acid to form metastable complexes (4-6). I t is a well-accepted fact that these ligand-avidin complexes dissociate upon exposure to biotin to form a very stable biotin-avidin complex. Figure 1 shows the association constants of a few biotin-related compounds in a ligand-avidin binding reaction. Schultz et al. suggested an affinity sensor for glucose using fluorescein-labeled dextran complexed with Con-A (7). The Con-A-immobilized fiber optic sensor is an convenient device for metabolites which are present in the M range.
On the other hand thyroxine was sensitively determined, in our preliminary study with membrane-bound thyroxine complexed with catalase-labeled antibody, in the concentration range from to lo4 g/mL (8). I t was the enzyme amplification technique that made the determination sensitive. However, the driving force of bioaffinity sensing is not necessarily clear, since the membrane-bound ligand and the determinant are the same molecules. The experiments of the present paper provide some further information on the bioaffinity sensor, especially from the viewpoint of driving force (bioaffinity difference). Figure 2 illustrates a schematic representation of the proposed bioaffinity sensor for biotin. The sensor is fabricated from a membrane, upon which a molecular complex between an analogue compound of biotin and enzyme-labeled avidin is prepared, and a transducer such as a Clark-type oxygen electrode. As far as biotin is concerned, HABA and lipoic acid are employed as the analogue compounds, whereas biotin and dethiobiotin are the determinants. Sensitization is to be attained by an enzyme amplification technique. EXPERIMENTAL S E C T I O N Materials. All chemicals were of analytical reagent grade, and all solutions were prepared with glass-distilled water. HABA (2-[ (4-hydroxyphenyl)azo]benzoic acid) and lipoic acid were purchased from Nakarai Chemicals (Kyoto). Water-soluble carbodiimide (l-cyclohexyl-3-(2-morpholinoethyl)carbodimide p-methyltoluenesulfonate) was obtained from Kokusan Kagaku (Tokyo). Cellulose triacetate was the product of Eastman Kodak (Rochester,NY).Avidin and ovalbumin (Grade 111)were obtained from Sigma (St.Louis, MO), catalase from Tokyo Kasei (Tokyo), and hydrogen peroxide (30%) from Santoku Chemical Ind. (Miyagi). 4-(Aminomethyl)-1,8-octanediamine was courteously supplied by Asahi Chemical Ind. (Tokyo). Preparation of Membrane-Bound Ligand. 4-(Aminomethyl)-1,8-octanediamine(2 mL) and 50% glutaraldehyde (400 WL)were added to cellulose triacetate (500 mg) dissolved in 5 mL of dichloromethane,and then cast on a glass plate. The aldehyde cross-linked 4-(aminomethyl)-1,8-octanediamine to polymerize throughout the stitch of cellulose triacetate. After drying at room temperature for a few days, the pink membrane was cut into small pieces, peeled off, and incubated for 1 h in 1% glutaraldehyde solution buffered with 0.1 M phosphate of pH 7 for 24 h. The membrane was then incubated in 1% ovalbumin solution for 1 h to immobilize the protein to the membrane surface. It is a well-known fact that biotinyl enzymes where biotin is bound to the t-amino groups of the enzymes are inhibited by avidin. Therefore, HABA (or lipoic acid) was immobilized with the method of carbodiimide conjugation to the membrane via the e-aminogroup of the albumin to decrease steric hindrance as little as possible. Two or three dozen membranes were incubated with 50 mg of HABA (or lipoic acid) and 88 mg of the carbodiimide. During coupling reaction the pH was controlled at 4.5. Finally, the polymer membranes were reduced with sodium borohydride under pH control at 7.0 with NaH2P04. The HABA (or lipoic acid) molecules not chemically but physically bound were washed out with 0.1 M carbonate buffer of pH 10 and then kept in storage in 1%ovalbumin solution at pH 7.0 to saturate the nonspecific binding sites. Figure 3 is a schematic representation of the membrane preparation. Preparation of Catalase-Labeled Avidin. Catalase (4 mg) in 1 mL of 0.1 M phosphate buffer (pH 7 ) was incubated with
0003-2700/85/0357-0496$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57,
-+BIOTIN
.
(KA=
NO.2, FEBRUARY 1985
497
/ CH,Cl,
101s
1
M)
-
i
Casting on a g l a s s plate
CDETHIOBIOTIN
( K A = 2 x 10”
I
M)
z .
Y
a
-
1
.+LIPOIC
.+
6
AC D
( 1 . 4 x 10 M ) HABA ( 1 . 7 x 105 M )
NaBH4
1 4
88
E E E
E E
~
0 @
Step
I
Determinant , 0 (Biotin) Binding protein, E (Avidin)
Step
I1
H20
Analog (Lipoate or HABA)
HH
.C-N
Enzyme
H
(Catalase)
Figure 2. Schematic representation of bioaffinity sensor. Molecular complex of low affinity is employed as a receptor. The first step is the biosensing of biotin, and the remaining molecular complex is sensitively detected by enzyme amplification.
50 WLof 2% glutaraldehyde for 10 min. Avidin (1.5 mg) previously dissolved in 1 mL of 0.1 M phosphate buffer (pH 7) was added to the catalase solution in a unimolar ratio and then incubated for another 20 min. After reduction of the Schiff bases between catalase and avidin with sodium borohydride, the mixture was concentrated with Sunwet IM-300 of Sanyo Kasei (Kyoto) and then chromatographed on a Sepharose CL-6B column. Assembly of Bioaffinity Sensor. The membrane-bound HAJ3A and catalase-labeled avidin underwent complex formation for 1 h in 0.1 M phosphate buffer (pH 7) containing 1% ovalbumin. The molecular-complex-formed membrane was tightly attached to a Clark-type oxygen electrode through a Teflon membrane. The diameter of a platinum electrode was 3 mm. Bioaffinity Sensing. The sensor was applied to the determination of biotin and dethiobiotin for 1 h at 37 “C. Every determinant was dissolved in 0.1 M phosphate buffer (pH 7) containing 1%ovalbumin. Ovalbumin was added to evaluate the interference from other substances in biological fluid. The sensor was then washed in 0.1 M phosphate buffer of pH 7. Into 45 mL of 0.1 M phosphate of pH 7 the sensor was placed and magnetically stirred to get a steady-state current at 30 “C. Five milliliters of 30 mM hydrogen peroxide was added to detect the catalase-labeled avidin that remained on the ligand-immobilizedmembrane. The catalase moiety produces many oxygen molecules through catalytic decomposition of hydrogen peroxide.
RESULTS Displacement Test for Avidin-HABA Complex with Biotin-Related Compounds. Addition of HABA in excess to avidin under conditions where almost all of it is bound gives a new absorption band a t 500 nm and a color change from yellow t o red (em increases from 600 to 34500). At the same time the 348-nm band of HABA almost disappeared ( 4 ) . These changes were reversed by the addition of 4 mol of biotin to 1 mol of avidin. The displacement of HABA by biotin-related substance is shown in Figure 4. The avidin (1mL of 5 pM avidin in 0.1 M phosphate buffer of pH 7) was first mixed with HABA. The
+
HABA
HABA
Flgure 3. Preparation of HABA-immobilized membrane: CTA, cellulose triacetate: AMODA, 4-(aminomethyl~l,8-octanediamine;GA, glutaraldehyde; HABA, 2-[(4-hydroxyphenyl)azo] benzoic acid; CDI, watersoluble carbodiimide; OA, ovalbumin.
Roleales of blotln-related mrpocnd / bln31ng slte
Flgure 4.
Displacement of HABA by biotin-related compound. HABA-avidin complex was substituted by biotin (0),dethiobiotin (O), and lipoic acid (A).The concentration of bound HABA was calculated by using the extinction coefficient of free and bound HABA from tEOO given in the text.
molar mixing ratio of HABA to one binding site of avidin was 2. Then the change in absorbance at 500 nm was measured after successive addition of a standard solution of biotin (15 mM phosphate of p H 7 ) with a micropipet till no further change occurred. Biotin analogues such as dethiobiotin and lipoic acid also displace HABA, t o an extent dependent on the binding constants. From this result, we come to the conclusion that such ligands as HABA and lipoate, which form metastable complexes with avidin, are employed. Fabrication of Bioaffinity Sensor. A cellulose triacetate membrane blended with a copolymer of 4-(aminomethyl)l&octanediamine and glutaraldehyde adsorbed a great deal
498
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
iI
A
I
63
-
3
-
4
0 I
2 4
0.2
1
0
0
10
20
30
0
40
of HABA; however, most of the adsorbed HABA was removed with thorough washings with 0.1 M carbonate of pH 10. Specroscopic study in the UV region showed that 3 fimol of HABA was covalently immobilized to a membrane through the ovalbumin bound to the membrane surface. Figure 5 illustrates the Sepharose CL-6B column chromatogram of the catalase-labeled avidin. Every fraction ( 2 mL) was monitored at 280 nm for the protein moieties of catalase and avidin and 406 nm for the heme moiety of catalase. The first and second peaks in the chromatogram were ascribed to catalase-labeled avidin. The second peak (from fraction 16 to 25) was concentrated to 4 mL with Sunwet IM-300. The catalase/avidin molar ratio of the conjugate was estimated for the second peak from the absorbance at 280 nm and 406 nm on the assumption that Ezsoof catalase and avidin, and Em of catalase were 14.6, 15.5, and 18.7, respectively (9). The approximate catalase/avidin molar ratio was 1.2. The binding capacity of catalase-labeled avidin was studied with HABA. A new absorption band at 500 nm was observed when the labeled avidin (0.2 mg/mL) was mixed with free HABA (14.6 X mol). The new absorption band at 500 nm was compared with that of native avidin complexed with free HABA. The estimated binding capacity of the avidin moiety was 20% of native avidin. Considerable binding capacity was lost after conjugation. However, approximately 80% of the labeled avidin seemed to bind HABA immobilized on the membrane, since avidin is a tetrameric protein. The absorption band disappeared immediately when free biotin was added to the avidin-HABA complex. Also the catalase activity in the conjugate was estimated to be 40% of free catalase with a spectroscopic method ( I O ) . The membrane-bound HABA was incubated at 37 "C in a solution containing 0.66 mg of catalase-labeled avidin and 10 mg of ovalbumin per 1 mL. The complex formation, Le., receptor preparation, terminated within 60 min. The HABA-immobilized membrane on which the molecular complex of low affinity was formed was tightly attached to a Clark-type oxygen electrode through a Teflon membrane. A shematic diagram of the proposed bioaffinity sensor is shown in Figure 2. Determination of Biotin and Dethiobiotin by the Bioaffinity Sensor. The time course of molecular recognition with the proposed bioaffinity sensor was studied. The sensor was immersed in a biotin solution (loe5g/mL) in the presence of 1%ovalbumin at 37 "C to observe the outlook of molecular recognition. Either HABA or lipoic acid was used as an analogue compound to be immobilized to the membrane. After the biosensing, the sensor was transferred to the measuring medium. The change in sensor output upon addition of hydrogen peroxide was recorded. Figure 6 illustrates the relation between the incubation time and the change in sensor output. The current change was
3115
€0
The for biotin reamitim ( min )
F r a c t i o n number
Flgure 5. Fractionation of catalase-labeled avidin. Every fraction (2 mL) was monitored at 280 nm (0)and 406 nm ( 0 ) .
15
Flgure 6. Time course of molecular recognition of biotin with membrane-bound HABA complexed with catalase-labeled avidin. Change in sensor output upon H 2 0 2 addition is shown in the inset figure (step I1 of Figure 2). Sensing times were 0 min (-), 2 min (- - -), 5 min (-). Hydrogen peroxide was added at the time indicated with an arrow.
i
s
v1 W
.-c
-0 L O
10-9
10-8
10-7
10-6
10-5
Biotin ( s/mL ) Flgure 7. Standard curve for biotin with bioaffinity sensor. The molecular complex of low affinity, membrane-bound HABA complexed with catalase-labeled avidin, was used as a receptor.
caused by the molecular complex receptor that remained on the sensor. Biotin recognition with the receptor (step I of Figure 2) finished within 10 min after the start of biosensing. The measurement of the remained receptor (step I1 of Figure 2) finished within 1 min as shown in Figure 6 (inset). Similar outlook of biosensing was observed with membrane-bound lipoate complexed with the labeled avidin. The catalase-labeled avidin adsorbed was not fully dissociated even in the presence of excess biotin. Approximately half of the receptor was undissociated. The HABA-immobilized membrane binds the labeled avidins by complex formation (specific binding) and adsorbs the labeled avidhs on its nonspecific binding sites. The labeled avidin, specifically bound to membrane-immobilized HABA (or lipoic acid), is expected to easily dissociate in the presence of excess biotin, whereas the nonspecifically adsorbed avidin is not. The response time of the proposed bioaffinity sensor was no longer than 10 min, although biosensing time was here set to 60 min. After biosensing at 37 "C for 60 min, the sensor was washed three times with 0.1 M phosphate buffer of pH 7. The sensor was transferred to a magnetically stirred medium buffered with 0.1 M phosphate of pH 7. After the oxygen-reducing current reached a steady state, 5 mL of 30 mM hydrogen peroxide prepared in 0.1 M phosphate buffer of pH 7 was quickly injected to make the remained molecular complex receptor detected. The change in sensor output promptly increased and reached a constant value within 1 min. The first result is shown in Figure 7 when biotin was determined with the bioaffinity sensor equipped with the membranebound HABA complexed with the labeled avidin. The change
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
499
Table I. Fundamental Characteristics of Bioaffinity Sensor
determinant analog
affinity
coverable
ratio
range, g/mL
biotin HABA 5.8 x 109 10-9-10-7 biotin lipoate 7.0 X lo8 5 X lO-lO-5 dethiobiotin HABA 1.2 X lo7 10-9-10-7
dissociable ratio, % 70
X
lo-@
50 45
in sensor output decreased as the concentration of biotin was increased. The determinable biotin was in the concentration range from IO+ (90% response) to io-' (10% response) g/mL. The midpoint concentration was lo-@g/mL. The standard deviation is approximately f l l %a t the midpoint concentration (n = 10). After the determination, the bioaffinity sensor was reimmersed in the catalase-avidin conjugate solution to prepare the molecular complex in the way mentioned above. The sensor was repeatedly used when the molecular complex was reprepared in the conjugate solution. It was not the stability of the membrane but that of the conjugate which limited the life of the sensor. So long as the catalase-labeled avidin is stable, the membrane was repeatedly used as membrane receptor for the biotin-related molecules. Figure 8 shows the correlation between dethiobiotin and sensor output obtained with the sensor of which receptor was the membrane-bound HABA complexed with the labeled avidin. The 90 and 10% response levels in the curve are and g/mL, respectively, whereas the midpoint biotin concentration was g/mL. The standard deviation is g/mL. approximately f15% a t Figure 9 is also a calibration curve for biotin with the bioaffinity sensor. The receptor was the membrane-bound lipoic acid complexed with the labeled avidin. The coverable range for biotin was from 5 x 10-lo(90% response) to 5 x (10% response) g/mL. The midpoint concentration was 2 X g/mL.
HABA from native avidin by the biotin terminates at the very moment of biotin addition since the bioaffinity difference is so great (see Table I). On the other hand, both the complex formation of membrane-bound HABA with the labeled avidin and the displacement of the avidin from the membrane-bound HABA by the biotin became much slower as shown in Figure 5. In all combinations studied above, perfect release of the labeled avidin from the membrane seems to be difficult even in the presence of an excess amount of biotin due to the presence of nonspecific adsorption of the labeled avidin. Table I shows the characteristics of the bioaffinity sensor in terms of bioaffinity difference (affinity ratio), coverable range, and the extent of dissociation. From these results, one may naturally come to the conclusion that the bioaffinity difference between analogue molecule and the determinant molecule does not so much depend on the sensitivity as the extent of dissociation. However, our preliminary experiment on the bioaffinity determination of lipoic acid with the membrane-bound HABA complexed with the labeled avidin does not give high sensitivity. Therefore, in the above three combinations where bioaffinity difference between an analog molecule and a determinant is greater than lo7,the sensing apparently does not correlate to the bioaffinity ratio, since the equilibrium lies so far to the right (stable complex formation). Comparing the time course of receptor preparation with that of receptor dissociation, one can understand that there are two binding states, i.e., specific and nonspecific bindings during the receptor preparation, and that one of them, specifically adsorbed complex, works a t the biosensing process. The disparity between the time required for receptor preparation and that for the biosensing is primarily due to the existence of nonspecific adsorption during the preparation of the molecular complex receptor. The role of ovalbumin was to decrease the number of nonspecific binding sites and to evaluate the interference in biological fluid. Bovine serum albumin (1%)was also found effective in the same manner. As far as the biosensing process is concerned, only the specifically bound avidin plays a key role. Consequently, further improvements of specific adsorption and desorption should be taken into consideration by controlling nonspecific binding as negligible as possible. Although a few points remain still to be improved, the present sensitive sensing concept based on bioaffinity difference and chemical amplification is a unique approach to the recognition of many a small molecule of biochemical importance. Besides the electrode-based approach mentioned here, the authors have recently shown that a photon counter-based bioaffinity sensor is effective in the sensitive determination of insulin (11). One further point requires emphasis. Physiologically active substances such as hormones and vitamins, which are susceptible to degradation during chemical modification, are easily determined, since the sensor does not require labeled determinant. Registry No. HABA, 1634-82-8;biotin, 58-85-5; dethiobiotin, 533-48-2; lipoic acid, 62-46-4.
DISCUSSION The complex formation of native avidin with free ligand such as HABA occurs instantly, and the displacement of free
(1) Berson, S.A,; Yalow, R. S. Clin. Chim. Acta 1968, 22, 51-69. (2) Jaffe, B. M.; B e h r m a n , H. R. "Methods of Hormone Radioimmunoassay"; Academic Press: New York, 1974; pp 473-476.
-0L O
10-9
10-8
10-7
Dethiobiotin
10-6 (
10-5
g/mL )
Flgure 8. Standard curve for dethiobiotin with bioaffinity sensor. The molecular complex, membrane-bound HABA complexed with catalase-labeled avidin, was taken as a dissociable receptor.
O
10-9
10-8
Biotin
10-7 (
10-6
10-5
g/mL )
Flgure 9. Standard curve for biotin with bioaffinity sensor. The low affinity molecular complex, membrane-bound lipoate complexed with catalase-labeled avidin, was employed as a receptor for biotin recognition.
LITERATURE CITED
500
Anal. Chem. 1985, 57,500-504
(3) Engvall, E. I n "Methods in Enzymology"; Van Vunakis, H., Langone, J. J., Eds.; Academic Press: New York, 1980; Vol. 70, pp 419-439. (4) Green, N. M. Biochem. J. 1965, 9 4 , 23c-24c. (5) Green, N. M. Biochem. J. 1986, 101, 774-789. (6) Green. N. M. I n "Methods in Enzvmoloav": McCormich. D. B.. Wrioht. L. D.. Eds.; Academic Press: New York:.1970; Vol. 18A, pp 414-434. (7) Schultz, J. s.;Sims, G. Biotechnoi. Bioeng. symp.1979, 9 ,65-91. (8) Ikariyama, Y.: Aizawa, M. R o c . 2nd Sensor Symp. 1982. 97-100
(9) Yamakawa, T., Ed. "Data Book of Biochemistry": Tokyo Kagaku Dojin: Tokyo, 1980; Voi. 1. pp 94 and 100. (10) Beers, R. F., Jr.; Sizer, I . W. J. Bioi. Chem. 1952, 195, 133-140. (11) Ikariyama, Y.; Aizawa, M. Proc. 3rdSensor Symp. 1983, 18-20.
RECEIVED for review August 22, 1984. Resubmitted October 30, 1984. Accepted October 30, 1984.
Determination of Oxide in Aluminum Chloride-Sodium Chloride Melts via Electrochemical Methods T. M. Laher, L. E. McCurry, and Gleb Mamantov*
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600
The use of TaCI, as a probe solute for the differential pulse voltammetric determination of oxide In AICI,-NaCI,,,, melts has been successfully demonstrated. This method makes use of the equilibrium reactlon TaCI,AIOCI,- F= TaOCI,- 4AICI,-. The analogous TI( I V ) method, used previously for basic AICI,-BPC melts, was found not to be applicable to the AICI,-NaCI melt. The equilibrium constant for the above reaction was determined to be (1.9 f 0.4) X l o 5 at 200 'C. I n addltlon to the Ta(V) method, an electrochemlcal cell employlng a P-alumlna membrane was shown to be useful In the potentiometric determlnatlon of oxlde In AICI,-NaCl,,, melts.
+
In an earlier study, McCurry (7) investigated the electrochemical behavior of Ta(V) in A1C13-NaCl,a,d melts as a function of oxide concentration in the melt. At low oxide concentration only a single voltammetric peak a t +0.65 V vs. Al(III)/Al in AIC1,-NaCLd melt was observed. The electrode reaction proposed for this process is given in eq 1. As the TaC16- -k e-
0003-2700/85/0357-0500$01.50/0
TaC1,'-
(1)
oxide content of the melt was increased, a second voltammetric peak at +0.45 V was observed, with the peak height increasing with increasing oxide concentration. The second process was ascribed to the following reaction TaOC1,-
Several attempts to determined the oxide content of chloroaluminate melts via electrochemical, spectroscopic, and other physical methods have been reported in the literature. For example, Tremillon et al. (1)proposed the use of a Ni/NiO electrode for the determination of oxide in AlC1,-NaCl melts. Later studies showed that the potential of this electrode is dependent on the chloride ion rather than the oxide ion activity ( 2 ) . Berg et al. (3) recently described a method for the determination of oxide in AlC13-NaC1 melts in which the oxide content of the melt is determined by difference of the weighed amount of AlCl, in the melt and that determined via potentiometric measurements. Various electrochemical studies have shown that the behavior of Nb(V) ( 4 ) ,Mo(V) (5),Ti(1V) (6), and Ta(V) (7) in chloroaluminate melts is dependent on the oxide concentration present in these melts. Osteryoung and co-workers (6,8) devised a voltammetric titration technique for the determination of oxide in the chloride ion rich (basic) room temperature aluminum chloride-N-(n-buty1)pyridinium chloride (AlCl,-BPC) melt using TiC14 as a probe solute. When Ti(1V) was added to the 0.8:l.O AlC1,-BPC melt which contained oxide, the species Tic&'- and TiOC1,'- were found to exist in equilibrium. The chloro complex could be reduced to Tic&-a t -0.35 V vs. A1 in 2.0:l.O AlCl,-BPC, while the oxychloro complex was reduced to Tic&,- at -0.77 V. From normal pulse voltammetric measurements of the reduction of Ti(1V) to Ti(II1) as a function of Ti(1V) concentration, the initial oxide concentration of basic AlCl,-BPC melts could be determined. Because of many similarities between the basic AlCl,-BPC melt and the AlC13-NaClsad melt, the possibility of using the above method for the determination of oxide in the basic AlC1,-NaCl melt was briefly explored. However, these attempts failed because TiC14, when added to AlC1,NaClSatdmelts, was found to be quite volatile with the result that the total Ti(1V) concentration in the melt rapidly decreased.
-
+ 2c1- + e-
-
TaCls2-
+ 0'-
(2)
Two other reduction peaks were also observed at +0.23 V and +0.15 V; these peaks were assigned to the further reduction of TaC&'- to lower oxidation states of tantalum. This type of behavior was also noted earlier by Ting ( 4 ) for Nb(V) in the AlC13-NaCLa melt in which the Nb(V) oxychloro species was found to be stable. This behavior is similar to the behavior of Ti(1V) in AlC1,-BPC (6,8). Furthermore, TaCl, is a relatively nonvolatile solid at typical working temperatures (175-200 "C) for A1C13-NaCl,a,d melts. Hence, TaCl, was believed to be a good probe solute for oxide determinations in AlCl,-NaCl,,d melts. Tremillon and co-workers (9) successfully employed an yttria-stabilized zirconia membrane electrode as an oxide ion selective electrode in the NaC1-KCl eutectic. However, attempts to use this electrode as an oxide indicator electrode in A1C13-NaCLd melt failed (10). Instead, this electrode was found to be chloride-ion selective in this melt. At present no known oxide ion selective electrodes for use in chloroaluminate melts exist. The exact nature of the oxide species in chloroaluminate melts has been the subject of much debate. The formula for the oxide species in basic chloroaluminate melts has been assigned in most of the reported literature as A10C12- (11). However, in a very recent study of the phase diagram of the A1C13-NaC1 system and the effect of oxide impurities on the freezing point depression of the melt, Berg et al. (3)proposed that the aluminum oxychloro species exists as a solvated dimer, A1302C&-.Their results did not rule out the possibility that this solvated dimer species was in equilibrium with other aluminum oxychloro species, most notably Al,OC&'- (solvated AlOClZ-).
EXPERIMENTAL SECTION Drybox System, Because of the air- and moisture-sensitive nature of the compounds used, all handling of materials and
6 1985 American Chemical Society
